Tuberculosis is the leading treatable infectious cause of death worldwide. A resurgence of tuberculosis has been observed in the United States since 1985, despite decades of public health efforts aimed at eradication. The causative agent in tuberculosis is Mycobacterium tuberculosis (MTb). Alarmingly, multiply drug resistant strains of Mycobacterium tuberculosis (MDRTb) have recently emerged and present a new and serious threat to health. In addition infection with another group of Mycobacteria, Mycobacterium avium complex (MAC) is a recent and dramatically growing health threat. MAC has been recognized to cause clinically significant disease in HIV-1 infected and other immunocompromised individuals. Disseminated MAC infection is estimated to be present in 17-28% of AIDS patients (Young, et al., Rev. Infect. Dis., 8: 1024-1033 (1986)) and this is thought to be an underestimate since 47-53% of AIDs patients have MAC detected at autopsy (Wallace et al. Chest, 93:926-932 (1988)).
Diagnosis of pulmonary tuberculosis (Tb) has traditionally been based on a constellation of symptoms elicited by medical history and supported by an abnormal physical exam and chest x-ray, a positive tuberculin skin test reaction, observation of mycobacteria in patient derived specimens, clinical response to anti-mycobacterial therapy, and/or recovery of Mycobacterium tuberculosis (MTb) by culture. In contrast, because the clinical symptoms of MAC are nonspecific, diagnosis of MAC infection is dependent on the recovery of MAC from culture of patient-derived specimens by the clinical microbiology laboratory.
In the past, laboratory based detection of mycobacteria in a wide variety of patient-derived specimens has been limited to either: i) direct detection, or ii) recovery by microbiological culture. Direct detection by use of a variety of acid fast stains (i.e., Kinyoun, modified Kinyoun, Fite, auramine-rhodamine, etc.), has a number of disadvantages. First, the sensitivity of direct detection is estimated to be only 30%-75% when compared to the ability to recover mycobacteria from culture. Second, detection is highly dependent on the expertise of the observer and the amount of time spent examining the stained preparation. Third, if bacilli are detected by acid fast stains, species identification is not possible.
In contrast to direct detection methods, there have been significant advances over the past decade in optimizing culture systems for the recovery of mycobacteria. Use of radiometric culture systems has dramatically shortened the length of time necessary to recover mycobacteria to a minimum of 7 days (as compared to 2-6 weeks for nonradiometric mycobacterial culture systems). With radiometric culture systems, only an additional 7 days are required to determine antimicrobial susceptibility patterns (as compared with the 2-6 weeks required for traditional mycobacteria antimicrobial susceptibility testing). The development of commercially available chemiluminescent probes for species-specific DNA (based on MTb-unique 16S rRNA sequences) has dramatically shortened to 4 hours the time necessary for species typing once mycobacteria are recovered from culture (as compared to 2-4 weeks for traditional mycobacterial species typing using biochemical tests). Despite this progress, culture methods still require considerable time (i.e., the initial 7 days to recover the mycobacteria from culture) to yield results.
Newer technologies have recently been used to increase the sensitivity and decrease the subjectivity of direct detection methods. The polymerase chain reaction (PCR) has been intensely investigated as a method for direct detection of mycobacteria in respiratory specimens. Unfortunately, the technology, expertise, physical environment requirements, and reagent costs of performing PCR currently far outweigh those necessary for the performance and interpretation of an acid fast stained smear. Furthermore, at the present state of development, PCR tests result in an unacceptably high rate of false positive results for specimens lacking mycobacteria. (See, e.g., Noordhoek et al., New Engl. J. Med. 329:2036 (1993) and Noordhoek et al., J. Clin. Microbiol. 32:277-284 (1994)).
Thus, the existing methods for detection of Mycobacteria suffer from lack of reliability, technical difficulty, lack of sensitivity, or extended time periods required for culture. The resurgence of Tb and the emergence of MDRTb and MAC have emphasized the necessity for methods allowing rapid, accurate and convenient detection, species identification, and susceptibility testing of Mycobacteria.